ANALYTICAL CHEMISTRY
1142 be washed. The remainder of the lanthanum-140 is not lost but stays with the barium-140. The quantitativeness of precipitation of barium nitrate in 80% nitric acid was determined by analyzing the filtrates for barium140 by the method of differential decay (6). The average barium concentration in the SO% nitric acid filtrates was 0.01 mg. of barium per milliliter. Since the total volume was 5 ml. and about 250 mg. of barium carrier was used, the amount of barium-140 remaining in the filtrate as a radioactive impurity in the lanthanum-140 was found to be approximately 0.03%. The recommended procedure is particularly effective for preparing carrier-free lanthanum-140 because only strontium, barium, radium, and lead form insoluble nitrates in SO% nitric acid ( 2 1 ) . Hence, the lanthanum-140 which grows in after the first milking need be separated only from these elements and their daughter activities. Radium and lead were both found to be absent, as the alpha activity in the lanthanum-140 mounts did not differ significantly from background. (All radioactive isotopes of lead and radium have alpha emitters in their decay chains.) The quantity of barium has been shown to be very log,, and the same is probably true for strontium (21). However, if exceptional purity is required ( 7 ) , the strontium activity can be separated by using a double barium chromate precipitation from homogeneous solution (16). The preparation can be carried out rapidly. The lanthanum140 can be easily recovered from the 80% nitric acid filtrate because of the very small volumes of solutions used. The entire procedure, including separation and recovery, requires less than 2 hours from the time a t which the separation from barium-140 is initiated. LITERATURE CITED
(1) Engelkemeir, D. W., Freedman, M. S., Glendenin, L. E., and Metcalf, R. P., “Characteristics of 12.8d Ba140,” Natl. Nuclear Energy Ser., IV-9, pp. 1104-07, Xew York, McGraw-Hill Book Co., Inc., 1951. (2) Greene, C . H., J . Am. Chem. Soc., 59, 1186 (1937).
(3) Gruen, D. M., Koehler, W. C., and Kata. J. J., Ibid.. 73, 1475, (1951). (4)
(5) (6) (7) (8) (9) (10)
Hillebrand, W.F., Lundell, G. E. F., Bright. H. h.,and Hoffman, J. I., “-4pplied Inorganic Analysis,” 2nd ed., p. 560, Kew York, John Wiley & Sons, 1953. Katcoff, S., Leary, J. B . , Walsh, K. d.,Elmer, R. d.,Goldsmith, S. S., Hall, L. D., Newbury, E. G., Povelites, J. J.. and Waddell, J. s., J . Chem. Phys., 17, 421 (1949). Kirby, H. W., ANAL.CHEM.,24, 1678 (1952). Kirby, H. W., and Salutsky, M. L., Phys. Rec., 9 3 , 1051 (1954). McCoy, H. N., J . Am. Chem. Soc., 58, 1577 (1936). Moeller, T., and Brantley, J. C., h s a ~CHmf., . 22, 433 (1950). Overstreet, R.. Jacobson, L., Scott, K., and Fisher, H., “Radiolanthanum (La19 ,” U. S. dtomic Energy Commission,
MDDC-1142A (1943). (11) Pagel, H. A., and Brinton, P. H. M.-P., J . Am. Chern. Soe., 5 1 , 4 3 (1929). (12) Quill, L. L., and Robey. R. F., Ibid., 59, 2591 (1937). (13) Quill, L. L., and Salutsky, 11.L., ANAL.CHEM.. submitted for
publication.
(14) Rodden, C. J., J . Research .YatZ. Bur. Standards, 26, 557 (1941). (15) Ibid.. 28. 265 (1942). (16) Salutsky, 11.L., Stites, J. G., and Nartin A. W., ANAL.CHEM., 25, 1677 (1953). (17) Schubert, J., and Richter, J. W.,“Studies on the Barium Citrate I
~I
Complex in Ammonium Chloride by the Ion Exchange Ilethod,” U. S. Atomic Energy Commission, AECD-1986 (declassified 1948). (18) Schweiteer, G. K., and Jackson, W. M.,J . 4 m . Chern. SOC.,74,
4178 (1952). (19) Tompkins, E. R., Khym, J. X., and Cohn, W. E., Ibid., 6 9 , 2769 (1947). (20) Wahl, A. C., and Bonner, S . A , , “Radioactivity ripplied to Chemistry,” pp. 93-101, 354-92, Sew York, John Kiley & Sons, 1951. (21) Willard, H. H., and Goodspeed, E. W,, IND.ESG. CHEM., A N A L . ED.,8, 414 (1936). (22) Willard, H. H., and Young, P.. J . A m . Chem. Soc., 50, 1379 (1928). RECEIVED for review October 28, 1953. Accepted February 1 7 , 1954. Abstracted in part from MLM-889, “Preparation of Carrier-Free Lanthanum140,” Mound Laboratory, Miamisburg, Ohio, July 31, 1953. N o u n d Laboratory is operated by Monsanto Chemical Co. under Atoinic Energy Commission Contract .4T-33-1-GEZT-53.
Analytical Total Acid Hydrolysis of Dextrans R. J. DIMLER, H. A. DAVIS, G. J. GILL, and C. E. RlST Northern U t i l i z a t i o n Research Branch, Agricultural Research Service, U. S. D e p a r t m e n t o f Agriculture, Peoria, 111.
Information was needed on optimum conditions for complete acid hydrolysis of dextran which could be used either in determining the concentration of dextran solutions or in liberating the constituent monosaccharide units for identification. Studies of the hydrolysis of dextran and destruction of D-glucose under a limited range of hydrolysis conditions showed that, within the limitations of the measurements, essentially the same maximum yield of reducing sugars and concurrent loss of D-glucose were obtained with 2 and 4 N sulfuric acid and 1 and 2 N hydrochloric acid at 100” C. and 1N sulfuric acid at 120” C., although the point of maximum yield was attained in periods of time ranging from 50 to 180 minutes. -4bout twice the normality of sulfuric acid was required to give the same results as hydrochloric acid in terms of both time of completion of hydrolysis and rate of destruction of D-glucose. A standardized procedure is given for analytical hydrolysis employing a 0.5% solution of dextran in 4.Nsulfuric acid heated at 100’ C. for 75 minutes, the reducing power
being corrected for an approximately 4% loss of D-glucose during hydrolysis. The applicability of the procedure to different types of dextran is demonstrated. Other sets of conditions for the analytical acid hydrolysis of dextrans can be selected on the basis of these studies.
T
HE synthesis of polysaccharides by the action of microorganisms, or enzymes therefrom, has assumed increasing importance in recent years. One group of such polysaccharides, the dextrans, has been given considerable attention as a source of blood volumeexpander products. The dextrans are polymers of D-glucose and have been produced from sucrose and from starch dextrins by the use of such microorganisms as Leuconostoc mesenteroides. Some of the microorganisms also are capable of producing fructose polymers, the levans. The total acid hydrolysis of dextrans has been used as a step in the determination of the amount of polysaccharide in solution aa well as in its characterization in part by identification of the con-
V O L U M E 2 6 , NO. 7, J U L Y 1 9 5 4 Ytituent monosaccharide units as D-glucose. Because of the importance of both of these applications in connection with a n existing research program on dextran as a blood volume-expander material, a study was made of the total acid hydrolysis of dextrans. .4 limited amount of information has been published on the acid hydrolysis of dextran. In general a variety of hydrolysis conditions has been employed, some of which seem quite unrelated. Thus, for the quantitative measurement of dextran in solution, hydrolysis periods of 5 t o 6 hours a t about 100" C. have been w e d with both 4% (6) and T.3% ( 1 5 ) sulfuric acid (approximately 0.8 and 1.5S, respectively), as well as with 1.0.4sulfuric acid ( 8 ) . For the characterization of dextrans by identification of the constituent monosaccharide unit as o-glucose, rcports have included hydrolvsis a t about 100" C. for 15 to 20 hours with both 0 . 1 S ( 2 4 ) and 1.0-V (1, 2 5 ) sulfuric acid, for 6 hours \$ith 4% (approximately 0.8.V) ( 5 ) )1 O N (9, 26), and appioximately 2 . 3 5 (4)sulfuric acid, and for 4 hours with 1.0-V (3) and 2.5% or about 0 . 5 s ( 7 ) sulfuric acid. Only one report of the use of hydrochloric acid for total hydrolysis of destran has heen noted, in nhich 3% (approximately 0 . 8 S ) acid \vas used at 100" C. with a hydrolysis time of 70 minutes in connection M ith the characterization of the crude polysaccharide ( 2 0 ) . In general no attention was given to possible extent of loss of D-glucose by destruction (or polymerization) during the period of hydrolysis, although such losses have been measured in connection with analytical hydrolysis of starch ( l 7 ' , 1 8 , d l ) . T h e studies reported here ~1ere designed to provide information on optimum conditions for analytical total acid hydrolysis of dextrans, including the effects of limited variations in hydrolysis conditions. Of particular interest was shortening the hydrolysis time to about 1 hour compared with the periods of 4 to 20 hours previously used. .4 comparison was made of hydrochloric acid 2 n d sulfuric acid relative to both the hydrolysis of dextran and to the loss of n-glucose (reducing power). For these studies the Somogyi reducing sugar method ( 2 3 ) was selected for folloning the course of reaction.
1143 The sensitivity of the reducing power determination to the salts formed on neutralization of the acids in the hydrolyzates was determined on samples of pure D-glucose. Sodium chloride had no effect a t the concentration of 1.2% which would be present M hen a hydrolyzate containing 2N hydrochloric acid is neutralized and diluted to a sugar concentration of about 0.5 mg. per ml. Sodium sulfate, on the other hand, caused a lowering of the glucose factor (milligrams of glucose per milliliter of 0.0055A' sodium thiosulfate) by about 2% when present in a concentration of 2.8% (equivalent to neutralization and dilution of a hydrolyzate containing 4-\-sulfuric acid). To eliminate any salt effects in the studies of hydrolysis, the unheated glucos+acid solution in each series was used for the determination of the glucose factor. Equipment. Most of the hydrolyses were carried out in thinwalled tantalum tubes, 0.5 mm. in wall thickness, 20 mm. in outside diameter, and 100 mm. long. The open end was flanged and fitted against a tantalum-lined cap held in place by a heavy steel ring-nut. A light layer of silicone stopcock grease was needed to secure the seal against leakage. A removable rack was used t o hold nine tantalum tubes vertically in the thermostated bath so that the liquid (ethylene glycol) level was just below the flanges. For later experiments use was made of 25 X 200 mm. borosilicate glass test tubes covered with loose-fitting glass thimbles, as used for the Somogyi reducing power determinations. There was no detectable loss of volume by evaporation a t 100" C. from these tubes during the heating periods of up to 6 hours. Experimental Hydrolyses. Solutions containing 0.45y0 dextran or 0.5y0 D-glucose (theoretical yield on hydrolysis of the dextran) in acid were prepared by weighing the required amount of carbohydrate into a volumetric flask, dissolving the sample in water, adding the appropriate quantity and kind of acid, and diluting to volume. I n five of the tantalum tubes were placed 20-ml. aliquots of the dextran-acid solution, while equivalent samples of the corre-
EXPERIMENTAL
Materials. The dextrans used in these studies Tvere well purified products prepared essentially as described by Jeanes et a!. ( 1 2 ) . For most of the trials a water-soluble dextran produced from sucrose by Leuconostoc mesenteroides SRRL B-512 was used; its analysis showed the presence of 0.02% nitrogen, 0.07% ash, and 0.01270 phosphorus. Periodate oxidation analysis (11) indicated that this dextran contained 94% 1,6'-glucosidic linkagej. T h e dextrans were stored and samples weighed in a constant-temperature and constant-humidity room, and the weights Mere corrected for the measured moisture contents of the dextrans. T h e n-glucose used was either Pfanstiehl C.P. or Bureau of Standards dextrose. Reducing Power Method. The Somogyi phosphate-buffered reagent ( 2 3 ) ,which incorporates a large amount of sodium sulfate to prevent back oxidation by air, was chosen as a rapid and convenient semimicromethod for reducing sugar determination. T h e iodometric version of the method, including iodate in the copper reagent, was used. T h e precision of the reducing power method in combination with the neutralization and dilution steps used for acid hydrolyzates was checked by determinations on unheated solutions containing 0.5% D-glucose in 4v7 sulfuric acid. d total of 37 aliquots were neutralized and analyzed, the reducing power being run in duplicate for 23 and singly for the other samples. T h e average deviation from the mean of all the samples was 3 ~ 0 . 6 % . Within each of the seven groups in which the samples were run the average deviation was only rt0.295, the largest deviation being 0.6%. Differences between groups, therefore, wire responsible for the larger over-all average deviation.
4 N H,SO,
2
5
IOO'C
H2S0, I 0 O " C
90 1 fi H,SO,
8 00
40
80
120°C 120
160
HYDROLYSIS
200
240
280
320
360
400
TIME, MINUTES
F i g u r e 1. Reducing Power, Calculated as D-Glucose, f r o m D e x t r a n a n d D-Glucose under Different Sets of Hydrolysis Conditions Broken curve. dextran Solid curve, D-glucose
ANALYTICAL CHEMISTRY
1144 sponding glucose-acid solution were placed in four others. The capped tubes were placed in the glycol bath or in boiling water simultaneously, or as nearly so as possible. I n the case of the glycol bath a t 120" C., a temperature drop to 115" C. occurred, but the bath had recovered fully in 5 to 6 minutes after introduction of the tubes. For the 100" C. bath, only a 2" C. drop was observed, and recovery to the constant (within 0.3" C.) value took less than 2 minutes. When the boiling water bath was used, boiling was interrupted but resumed in 2 to 3 minutes. At the selected time intervals single tubes (from the dextran or the glucose group or from each) were removed from the bath, chilled in cold water, and opened. The hydrolyzates were rinsed promptly into beakers containing slightly less than the theoretical amount of cold sodium hydroxide solution required to neutralize the acid. Neutralization to the phenolphthalein end point was completed by titration. Each neutralized hydrolyzate was diluted to 200 ml. (theoretically containing 0.50 mg. of glucose per ml.) and duplicate 5-ml. aliquots were taken for determination of the apparent glucose content by the Somogyi reducing power method. Curves showing the course of dextran hydrolysis and glucose destruction under five different sets of conditions are given in Figure 1. Nost of the points are averages of the results on two to four separate hydrolysis runs. The time a t which the maximum in the hydrolysis curve is reached and the reducing p o ~ i . recoveries a t that time are given in Table I.
Table I. Yield of Glucose from Dextran and D-Glucnse under Conditions for Maximum Dextran Hydrolysis'' Acid and Temp.,
c.
a
b
Time of Maximum, Min. 75 190 54 150 50
Reducing Power' from Dextran, D-glucose, 7c
"0
4N H2S04, 100 96.3 96.8 2 N HzSO4, 100 95.5 97.0 2 X HC1, 100 96.5 97.4 I N HC1. 100 96.8 97.5 1,V H2S06, 120 95.6 96.5 From curves Figure 1. By Somogyi'method ( B ) expressed , as D-glucose, 'Z of theoretical
The effect of polysaccharide structure on the recovery of reducing sugars under one set of optimum hydrolysis conditions was determined by making a number of single-tube hydrolysis runs on purified dextrans of widely different type from B-512, on n levan produced as a by-product of a B-512 enzymic synt h e w of dextran ( I d ) , and on a cornstarch sample. The yields of apparent glucose, measured by reducing power, after a 75-minute hydrolysis with 4N sulfuric acid a t 100" C. are given in Table 11, along with the corresponding data on a typical run Rith the B-512 dextran. Nature of Glucose Loss. Dextran hydrolyzates, deionized Kith an anion exchange resin (Duolite il-6) or by neutralization with silver carbonate or barium carbonate, were subjected to qualitative paper chromatography ( 1 4 ) . Aside from D-glUCOSC,
Table 11. Results of Hydrolysis of Different Polysaccharides by t h e Preferred Procedure
*
'I
d e
(75 minutes in 4 N H&04 at 100' c.) 1,6'Observed Linkages Reducing Present, Powera. Recovery b , Polysaccharide lo % 7% Dextran B-512 94 96.0 100.2 Dextran B-742 66 95.6 99.4 Dextran B-113QC 80 96.9 100.7 Dextran B-1255 91 96.8 100.6 Starch (corn) 96.0 99.8 Levan from B-512-Eb 26 ... By the Somogyi method ($a), expressed as D-glucose, % of theoretical Corrected for a loss of 3.8% D-glucose under hydrolysis conditions. Essentially insoluble in water. Remaining 96% of the linkages are a-l,4'-glucopyranosidio. A fructose polymer formed as a by-product in a n enzymic synthesis of B-512 dextran.
only a very small amount of material having the R , of a disaccharide such as isomaltose or gentiobiose and a bare trace of 3 slower moving saccharide could be detected. The apparent amounts of these additional sugars were not altered significantly by varying such factors as the acid used (2N hydrochloric or 4iY sulfuric acids) in the hydrolysis a t 100' C. or the hydrolysis time (from about 1 to 6 hours with either acid). When the hydrolyzates n-ere incompletely deionized, poor chromatograms were obtained because of the large amounts of sample (approximately 1 mg. of carbohydrate in a spot 3 to 5 mm. in diameter on the starting line) which had to be applied to the paper in order to detect the polymeric sugars on the chromatogram. Anthrone determinations (2, 22) of total carbohydrate on a solution of D-glucose in 4F sulfuric acid heated 6 hours a t 100" C. showed a carbohydrate content in good agreement with the glucose content measured by reducing power. These data confirm the observation by Lampitt et al. ( 1 ; ) that a t the 10%- concentrations of carbohydrate used for snnlgtical acid hydrolysis of polysaccharides, the loss of reducing power results primarily from destruction of D-glucose rather than its recombination or "reversion." DISCUSSION
.4n indication of the reproducibility of the over-all procedure in these studies has been obtained by inspection of the data used in preparing the curves in Figure 1. Of the 46 experimental points shown (not including the zero-time points), 43 are averages of the results from two to five separate hydrolysis runs. For these points, the average deviation of the replicate values from their mean m s +0.6%. The largest deviation from the mean was 1k1.470, except for three points which included valucs having xider deviation (glucose and dextran in 211' sulfuric arid for 360 minutes a t 100" C. and dextran in 1-V sulfuric acid foi 150 minutes a t 120" C.). The conclusions presented here, therefore, arc based, in general, on attaching significance to variations greater than about +0.5% between the values taken from the curves in Figure 1. The results of the studies of dextran hydrolysis and D-gluroie deqtruction are similar in many respects to those obtained in comparable studies of starch hydrolysis (17, 18, 21). Thus the reducing power of the dextran hydrolyzates rises to a maximum n hich is less than theoretical and then slowly decreases The curve for reducing pouer from o-glucose treated under the h l drolysis conditions is essentially linear (zero-order or early stages of first-order reaction) after the first few minutes. The dextran hydrolysis curves also are essentially linear beyond the maximum and nearly superimposed on the corresponding I)glucose curves. Therefore, a t and beyond the maximum a correction of the reducing power of the dextran hydrolyzate for the extent of loss of n-glucose under the same conditions gives the theoretical yield within the limitations of these measurement.. The maximum reducing power from dextran is obtained after a longer period of hydrolysis than for starch (Table 11)as would he expected from the slower rate of hydrolysis of a-1,6'- than a-l>-i'glucosidic linkages (IS, 16, $7). Comparison of Acids. At the point of maximum reducing poR-er in the hydrolysis of dextran the extent of destruction of Dglucose ~ a the 3 same (about 4%), within the limitations of the measurements, regardless of whether sulfuric or hydrochloric acid was used (Table I). .4t a given normality of acid it is true that sulfuric acid caused slower loss of D-glucose than did h\-drochloric acid (18, 19). This difference in rate of destruction, however, is balanced by the need for a longer hydrolysis time to reach the maximum in the dextran hydrolysis curve with sulfuric acid. Thus the maximum was reached in about 180 minutes with 2.V sulfuric acid while only about 50 minutes was required with 2-V7hydrochloric acid. The possibility of this type of relationship between polysaccharide hydrolysis and glucose destrucntion was not mentioned by Pirt and Whelan ( 2 1 )in their selection
V O L U M E 2 6 , NO. 7, J U L Y 1 9 5 4 of sulfuric acid for starch hydrolysis because of its slower action on D-glucose. I n general, for sulfuric acid a normality somewhat over twice that for hydrochloric acid is required to give essentially the same results in terms of both time of maximum reducing power in the dextran hydrolysis and the corresponding extent of loss of Dglucose (Figure 1 and Table I). Temperature of Hydrolysis. I n general, the use of a boiling water bath for heating the hydrolyzates is to be preferred because of convenience. HoMever, for the initial stages of the present studirs an ethylene glycol bath thermostated a t 100" =k 0.3" C. was used, together with thin-walled tantalum pressure tubes, to ensure quick heating of the samples to a reproducible temperature. Subsequent comparative studies using loosely capped glass tubes and a boiling water bath gave identical results, within the limitations of the measurements. Since the temperature of boiling water may vary, a comparison was made of the hydrolysis of dextran in a tantalum tube at 98" and 100" C. with 4 S sulfuric acid. .4t 75 minutes, when the reducing power of the 100" C. run had reached a maximum, the reducing power of the 98" C. hydrolyzate was lower by only about 1.0%. The latter run reached maximum reducing power in about 90 minutes. Therefore, within the limitations of the measurements in the preaent procedure, the probable temperature variations in a boiling water bath would have no significant effect on the results. Since some workers may wish to use temperatures higher than 100" C. in order to tiecrease the strength of acid required, limited studies were made with a hydrolysis temperature of 120" C. (Figure 1 and Table I ) . This higher temperature permitted the use of 1 S sulfuric acid for 30 minutes instead of the 4N sulfuric acid for 75 minutes used a t 100" C. The extent of loss of Dglucose a t the point of maximum reducing power of the hydrolyzate was only slightly lower than a t 100" C., the significance being uncertain in view of the limitations of the procedure. The use of a steam autoclave and glass test tubes, instead of a glycol bath and tantalum tubes, gave fairly consistent results for 120" C. hydrolysis. Difficulties were noted, however, in the accurate control of the temperature and in the measurement of the hydrolwis time with the equipment used for these trials. SUGGESTED PROCEDURE FOR DETERMIN 4TION O F DEXTR4N BY ACID HYDROLYSIS
To illustrate the application of these studies to the selection of conditions for the total hjrdrolysis of dextran, either for qualitative or quantitative determinations of dextran, the following procedure n-as adopted. Sulfuric acid was chosen because of its slight advantages of nonvolatility and ease of removal as barium sulfate in case a qualitative examination is to be made of the hydrolyzate. The use of 4Al'sulfuric acid provides a conveniently short hydrolysis time of about 75 minutes, yet the maximum in the hydrolysis curve is sufficiently broad that small errors or small changes in temperature will not change the results significantly. The boiling water bath, instead of a glycol bath, again is a matter of convenience and simplification of equipment requirements. The tubes used for the hydrolysis are the same kind as those used in the Somogyi determination of reducing sugars.
A sample containing about 0.1 gram of dextran is accurately weighed (=t0.2%) or measured into a 25 X 200 mm. borosilicate glass test tube, then is dissolved or dispersed in enough water to bring the liquid volume to 10.0 ml. Sulfuric acid, 10 ml. of S . O N , is added and mixed in thoroughly by swirling. The test tube, loosely capped with a flared glass thimble or similar closure, is immersed in a boiling water bath to a depth of 100 to 150 mm. If the sample has not dissolved before heating, the tube is removed from the bath a t 1- to 2-minute intervals, quickly swirled, and returned to the boiling water. This is repeated until no undissolved material remains, usually not over three times. A4ftera total heating time of 75 minutes, the tube is cooled (to below 20" C.) in a cold water bath and its contents are quickly
1145 poured and rinsed into a beaker containing 75 ml. of cold (below 10" C.), well-stirred l A r sodium hydroxide. The slightly acid solution which results is titrated to the phenolphthalein end point using 1N sodium hydroxide, then warmed to room temperature and diluted to 200 ml. in a volumetric flask of that capacity. Duplicate 5-ml. aliquots of this solution are taken for determination of reducing power, expressed as glucose, by the Somogyi method ( 2 3 ) . The reagent should be standardized against a standard glucose solution which is 0.2-V in sodium sulfate (5.7 mg. sodium sulfate per mg. of glucose). Calculation, from reducing power, expressed as dextrose in the 5 ml. taken for analysis:
% dextran in sample
=
mg. of glucose in 5 ml. X 200 j X 93.6 mg. of orig. sample
or Mg. dextran in sample
=
mg. of glucose in 5 ml. X
200 T
X 0.936
The factor 0.936 results from the fact that theoretically 0.90 gram of dextran yields 1.00 gram of glucose, but under the hydrolysis conditions used only 0.962 gram is obtained, so that the 0 90 factor for converting the glucose yield back to dextran is A 0.962' If other hydrolysis conditions are to be used, the factor will remain essentially the same as long as the hydrolysis is stopped at the time of maximum reducing power and the conditions are approximately in the ranges covered by the described studies The factor is determined conveniently by measuring the loss of reducing poiver of a sample of D-glucose under the hydrolysis conditions. Effect of Variations in Polysaccharide Structure. Dextrans are known to differ greatly in structure and properties, depending on the strain of microorganism used in their synthesis ( 1 8 ) . Thus the content of 1,6'-glucosidic linkages, as measured by formic acid production on periodate oxidation (11), has ranged from about 97 to 50% (IO). The non-l,6'-1inkages apparently may be 1,4'- , 1,3'-, or both. Since o/-1,4'-glycopyranoqidic linkages, a t least, are knoxm to be hydrolyzed more rapidly than a-1,6'glucopyranosidic linkages [starch vs. dextran (13, 16, 2 7 ) and maltose t.s. isomaltose ( 2 8 ) ]it, R-as nccessary to determine whether structural differences between dextrans would influence the results obtained under a standard set of hydrolysis conditions. The general applicability of the recommended hpdrolgsi.; procedure (0.5% dextran in 4 5 sulfuric acid hydrolyzed 75 minutes a t 100" C.) is demonstrated by the results in Table I1 for a representative selection of dextrans differing in content of 1,6'-linkages and physical properties. The recoveries, after application of the standard correction for loss of D-glucose, were 100 f 0.7%. The variations from theoretical recovery reflect the variability for the over-all procedure rather than differencw in the polysaccharides. The favorable insensitivity of the determination to differences in dextran structure undoubtedly is a result of the fact that for both dextran and starch (and probably for other polyglucosans) the reducing power curves for the polysaccharide and for Dglucose under the same conditions are essentially superimposed beyond the maximum in the hydrolysis curve. Under a given set of conditions, therefore, the correction factor for n-glucose destruction is applicable, regardless of the polyglucosan structure, as long as the maximum in hydrolysis has been reached or passed. This is illustrated by the results a i t h starch (Table 11). .41though the maximum reducing power had been reached in 30 minutes, the corrected yield a t 75 minutes, using the same calculation as for dextran, was quantitative [see also ( I ? ) ] . Since the 1,4'- and 1,3'-linkages in dextran probably are more easily hydrolyzed than the l,B'-linkages, all the dextran samples in Table I1 had either reached or passed the maximum during the 75-minute period of hydrolysis and the quantitative recoveries observed are readily accounted for. These hydrolysis conditions are not applicable to fructose polymers. Thus a levan obtained as a by-product in the enzymic synthesis of a B-512 dextran yielded only about one fourth of the theoretical reducing power when hydrolyzed under the condi-
1146
ANALYTICAL CHEMISTRY
tions suggested here. This low yield obviously was a result of extensive decomposition of the relatively acid-sensitive ketohexose since the hydrolyzate was very dark in color. I n conclusion, therefore, the concentration of dextran in solution can be determined by total acid hydrolysis, provided a correction is made for concurrent destruction of D-glucose. The method is not affected by differences in the structure and properties of the dextran. The total hydrolysis procedure, of course, is not a method for the specific determination of dextran in the presence of other polysaccharides-for example, starch gives the same yield of reducing power as dextran. I n the use of total acid hydrolysis as a step in the characterization of preparations thought to be dextrans, allowance must be made for the fact that an acid labile sugar such as fructose will be largely destroyed under the conditions of hydrolysis. Thus small amounts of combined fructose, present either as a levan contaminant or as units in a mixed polymer with D-glucose, may escape detection. T h e presence of larger amounts of fructose would be indicated b\darkening of the hydrolysis mixture and the detection of a t least traces of fructose in the hydrolyzate. ACKNOWLEDGxMENT
The authors wish to thank Carl S. Wise for assistance in the running of paper chromatograms and anthrone determinations and Allene Jeanes and Carl A. Kilham for samples of dextran and levan. LITERATURE CITED (1) Daker, W. D., and Stacey, hI., Biochem. J . (London), 32, 1946 (1938). (2) Dimler, R. J., Schaefer, W. C., Wise, C. S., and Rist, C . E., ANAL. CHEM., 24, 1411 (1952). (3) Forsyth, W. G. C . , and Webley, D. M.,Biochem. J . (London), 4 4 , 4 5 5 (1949).
Fowler, F. L., Buckland, I. K., Brauns, F., and Hibbert, H., Can. J . Research, B15, 486 (1937).
(4)
(5) Gronwall, A., and Ingelman, E., Acta PhysioL. Scand., 7, 97 (1944). (6) Ibid., 9, 1 (1945). (7) Hassid, W. Z., and Barker, H. A., J . Bid. Chem., 134, 163 (1940). (8) Hehre, E. J., Ibid., 1 6 3 , 2 2 1 (1946). (9) Hehre, E. J., and Sugg, J. Y . ,J . Ezptl. Med., 7 5 , 3 3 9 (1942). (10) Jeanes, Allene, Haynes, W. C., Wilham, C. A., Rankin, J . C. and Rist, C. E., Abstracts of Papers, p. 14A. 122nd Meeting of the . ~ M E R I C A NCHEMICAL SOCIETY, September 1952. (11) Jeanes, Allene, and Wilham, C. A., J . Am. Chem. Soc., 72, 2655 (1950). (12) Jeanes, Allene, Wilham, C. A , , and Miers, J. C., J . Biol. Chem., 176, 603 (1948). (13) Jeanes, Allene, Wilham, C. A., and LMiers,J. C., unpublished
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g.,
RECEIVED for review February 27, 1954. Accepted April 26, 19.54. Presented before the Division of Carbohydrate Chemistry at the 125th Lleeting of the VERICA CAS CHEMICAL SOCIETY, Kansas City, \Io., March 1984.
Radioactive Tracer Assay for Vitamin Btn and Other Cobalamins In Complex Mixtures F. A. BACHER, A. E. BOLEY, and
C. E. SHONK
Merck & Co., lnc., Rahway, N. J. \-itamin BIZcan be assayed in complex mixtures ranging from fermentation products to vitamin capsules, by the purification and concentration of the vitamin. Other cobalamins can be determined after conversion to vitamin B I ~ .Radioactive vitamin BIJis used as a tracer to determine recovery through the various extractions necessary for purification; the amount of purified vitamin BIZis determined spectrophotometrically. A combination of purification operations can be selected to fit each type of sample. Samples containing 100 y of vitamin Blz at concentrations as low as 0.1 y per ml. can be assayed. In a series of difficult assays a standard deviation of *4.3% was found.
T
HE availability of vitamin BIZ (cyanocobalamin) labeled
with cobalt-60 ( 2 , 4 ) has led to the development of methods which permit the determination of cyanocobalamin in mixtures from which its quantitative isolation is impossible. I n view of the wide application of these tracer methods, this paper stresses useful techniques rather than detailed analytical procedures.
The methods of extraction and purification, when applied t o a variety of fermentation products and other mixtures, yield aqueous solutions sufficiently pure for spectrophotometric determination of cyanocobalamin. Additional identification tests can be applied, although the combination of extractions usually required for adequate purification appears t o have high specificity for cyanocobalamin. While purity and identity of both tracer and pioduct are essential t o a tracer assay, adequate characterization can be achieved without isolation of crystalline cyanocobalamin, as the most telling criteria-Le., absorption spectrum ~ i i dbenzyl alcohol-JTater distribution-are applied in solution. .Is it is impracticable to make a suitable tracer for each of the large group of cobalamins, these materials can best be assayed by conversion t o cyanocobalamin and subsequent assay, using the radioactive cyanocobalamin tracer. The term “readily convertible” 18 applied here t o substances which can be converted t o cyanocobalamin by the procedure recommended below. An assay for total cobalamins can be made which includes cyanocobalamin plus readily convertible substances, expressed as cyanocobalamin. The conditions necessary for converjion t o cyanocobalamin depend on the nature of the sample. Although it is
